CN114317431A - Method for promoting differentiation of human hematopoietic stem/progenitor cells to erythroid line - Google Patents

Abstract

The invention discloses a method for promoting differentiation of human hematopoietic stem/progenitor cells to erythroid. The method is to provide a hypoxic environment for the culture of human hematopoietic stem/progenitor cells or a drug that mimics a hypoxic environment. The inventor discovers that the hypoxia has the function of accelerating erythroid differentiation and can overcome the defect of low differentiation efficiency in the prior art, the hypoxia is the microenvironment of various stem cells, the inventor discovers the function of the hypoxia in promoting erythroid differentiation in vitro for the first time, and the function can be applied to treating diseases such as severe wounds, hematopathy, immunodeficiency syndrome, malignant tumors and the like and is beneficial to relieving anemia symptoms of patients.

Description

A method for promoting erythroid differentiation of human hematopoietic stem/progenitor cells

technical field

The invention belongs to the technical field of stem cell differentiation, and in particular relates to a method for promoting the differentiation of human hematopoietic stem/progenitor cells to erythroid.

Background technique

Transfusion of red blood cell components is an important guarantee for the effective treatment of serious trauma, blood diseases, immunodeficiency syndromes, and malignant tumors. At present, blood products such as red blood cells used in clinical use are mainly derived from voluntary blood donation by donors; however, countries around the world are still faced with multiple challenges such as unbalanced supply and demand of blood products, poor blood compatibility, and contamination by pathogenic microorganisms. Therefore, finding a more adequate, safe and economical alternative source of blood in addition to voluntary blood donation by donors will greatly improve the current predicament. In recent years, the development of stem cell technology has gradually established the method of inducing hematopoietic stem/progenitor cells from human umbilical cord blood, bone marrow and peripheral blood to differentiate into red blood cells in vitro; hematopoietic stem/progenitor cells from umbilical cord blood and other sources have extremely strong The self-renewal and multi-directional differentiation potentials can follow the developmental rules of erythroid cells in vivo under specific culture conditions, and go through multiple developmental stages such as hematopoietic progenitor cells-erythroid progenitor cells-erythroid precursor cells (primary erythrocytes to late erythrocytes). , step-by-step directional differentiation to form denucleated red blood cells, which makes it possible to prepare a large number of blood components such as mature red blood cells for clinical use, and can provide a potential source of high-quality blood replacement to meet future clinical needs. However, the existing induction methods are far from meeting the requirements of clinical application in terms of erythrocyte differentiation efficiency.

The maintenance of self-renewal, proliferation and multi-directional differentiation potential of hematopoietic stem/progenitor cells requires a certain microenvironment. point. Different stem cells have different microenvironments, and hypoxia is a common component of many stem cell microenvironments. Hypoxia is also a microenvironment for HSPCs. The inventor's research found that the hypoxic microenvironment has an important regulatory effect on the self-renewal and multi-directional differentiation potential of hematopoietic stem/progenitor cells. The erythroid differentiation of hematopoietic stem/progenitor cells provides theoretical basis and technical support.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the shortcomings of the prior art, and to provide an induction method that can accelerate the differentiation of human hematopoietic stem/progenitor cells into erythroid.

A method for promoting the differentiation of human hematopoietic stem/progenitor cells to erythroid, the method is to provide a hypoxic environment for the culture of human hematopoietic stem/progenitor cells or a drug that simulates the hypoxic environment.

The hypoxic environment is where the oxygen content is less than 3%.

The medicine for simulating hypoxic environment is one or more of FG-4592, COCl ₂ , and deferoxamine.

The medicine for simulating hypoxic environment also contains one or more pharmaceutically acceptable excipients or carriers.

The adjuvant or carrier includes one or more of conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, and lubricants in the pharmaceutical field. kind.

The dosage form of the medicine simulating hypoxic environment is selected from tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral liquids, pills or injections.

The hematopoietic stem/progenitor cells are derived from umbilical cord blood, bone marrow or peripheral blood.

The application of the red blood cells obtained by the method in the preparation of medicines for the treatment of trauma, blood diseases, immunodeficiency syndromes, malignant tumors and anemia diseases.

The blood diseases mainly include: aplastic anemia, thalassemia, sickle cell anemia, myelodysplastic syndrome, purpura, hemolysis and the like.

The hypoxic conditions or chemical drugs provided by the present invention may only use hypoxia as an intervention condition, or may be used in combination with hypoxia and a chemical drug that simulates hypoxia, or may be used in combination with two or more other chemical drugs Promote erythroid differentiation in vitro.

Beneficial effects of the present invention: The present invention finds that hypoxia has the effect of accelerating erythroid differentiation, and can overcome the disadvantage of low differentiation efficiency in the prior art. Hypoxia is a microenvironment for a variety of stem cells. The inventors have discovered for the first time its role in promoting erythroid differentiation in vitro. This role can be applied to treat diseases such as severe trauma, blood diseases, immunodeficiency syndrome, and malignant tumors. Relieve the symptoms of anemia in patients.

Description of drawings

Figure 1 shows the mRNA expression levels of erythroid differentiation marker molecules γ-globin and β-globin at different time points in induced erythroid cells cultured under normoxia (Normoxia) or hypoxia (Hypoxia) conditions by qPCR.

Figure 2 shows the protein expression levels of γ-globin and β-globin at different time points in the induced erythroid cells cultured under normoxia (N) or hypoxia (H) conditions by Western blot method.

Figure 3 is a flow cytometry method to detect the changes in the ratio of CD71 ⁺ /CD235a ⁺ cells at different time points in the induced erythroid cells cultured under normoxia (Nor) or hypoxia (Hyp) conditions. .

Figure 4 shows the changes in cell morphology of induced erythroid cells obtained by Giemsa staining after being cultured under normoxia (Nor) or hypoxia (Hyp) conditions for different times.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

The carbon dioxide incubator used in the following examples was purchased from American Thermo Fisher, product model: 3131, serial number: 300158130; the carbon dioxide incubator used in the comparative example was purchased from American Thermo Fisher, product model: 3111, serial number: 300205034.

Example 1: Method for promoting erythroid differentiation of human umbilical cord blood-derived hematopoietic stem/progenitor cells

1. Low oxygen concentration setting

(1) The _O2 concentration of the carbon dioxide incubator used is set to 3%;

(2) Treatment of hematopoietic stem/progenitor cells: hematopoietic stem/progenitor cells were expanded under normoxia for 7 days after resuscitation, and then induced to differentiate into erythroid cells for 11 days under normoxia and hypoxia respectively;

(3) Control group (comparative example): The carbon dioxide incubator used in the control group is set to the oxygen concentration by default (that is, the atmospheric oxygen concentration, about 20%).

2. Culture of Hematopoietic Stem/Progenitor Cells

Expansion Medium (EM) contains: StemSpan SFEM Medium (Cat. No. 09650) and 50ng/ml rhSCF (Cat. No. 300-07), 100ng/ml rhTPO (Cat. No. 300-18), 100ng/ml rhFlt3-Ligand ( Cat. No. 300-19), 50ng/ml rhIL-3 (Cat. No. 200-03) and 100ng/ml rhIL-6 (Cat. No. 200-06). StemSpan SFEM medium was purchased from STEMCELL Technologies, Canada, and the above cytokines were purchased from PeproTech, USA.

Erythroid differentiation medium (EDM, namely EDM3) contains: IMDM medium (Sigma Aldrich, USA, product number: 16529) and 3U/ml rhEPO (PeproTech, 100-64), 2mM L-Glutamine (product number: G7513), 330μg /mlhuman holo-Transferrin (Item No.: T4132), 10μg/ml human insulin (Item No.: I9278), 2U/ml heparin (Item No.: 07980) and 5% inactivated human serum (Item No.: H3667). IMDM medium, L-Glutamine, human holo-Transferrin, human insulin and human serum were purchased from SigmaAldrich Company in the United States, rhEPO was purchased from PeproTech Company in the United States, and heparin was purchased from STEMCELL Technologies Company in Canada.

Phase I erythroid differentiation medium (EDM1) consists of EDM supplemented with 100 ng/ml rhSCF, 5 ng/ml rhIL-3 and 1 μM dexamethasone (Cat. No.: D4902). Dexamethasone was purchased from Sigma Aldrich, USA. Phase II erythroid differentiation medium (EDM2) consisted of EDM supplemented with 100 ng/ml rhSCF.

(1) Resuscitate the human umbilical cord blood-derived hematopoietic stem/progenitor cells (HSPCs) frozen at -80°C, sort the cells into culture wells, add an appropriate amount of pre-warmed expansion medium (EM), and put them at 37°C , 5% CO ₂ incubator for 7 days; fresh culture medium was changed on the 2nd day, and then the medium was changed every other day.

(2) On the 8th day after resuscitation, the expanded HSPCs were inoculated in a new culture dish, and the phase I induction medium (EDM1) was added to the cells in normoxia (normoxia, 20% O ₂ , control), respectively. The culture was continued for 7 days under conditions of hypoxia (hypoxia, 3% O ₂ , Example), the seeding density was 3×10 ⁵ cells/ml, and the medium was changed every 3 days.

(3) After 7 days of induction of differentiation, the phase II induction and differentiation medium (EDM2) was replaced, and the seeding density was 8×10 ⁵ cells/ml, and the conditions were continued in normoxia (normoxia, 20% O ₂ , comparative example) and hypoxia ( Partially denucleated erythroid cells were obtained by culturing for 4 days under the conditions of hypoxia, 3% O ₂ , example).

(4) After 11 days of induction, the phase III induction medium (EDM3) was replaced, and the culture was continued under normoxia (normoxia, 20% O ₂ , Comparative Example) and hypoxia (hypoxia, 3% O ₂ , Example) conditions A higher percentage of denucleated erythroid cells can be obtained.

Example 2: qPCR experiment

The obtained cells were collected and lysed at different time points according to conventional methods, and RNA was extracted and analyzed by qPCR to detect the mRNA expression changes of erythroid marker molecules γ-globin and β-globin. The primer sequences used in the experiment are shown in Table 1:

Table 1

The results are shown in Figure 1. When HSPCs were induced to differentiate, the expression of erythroid marker molecules γ-globin and β-globin at the mRNA level gradually increased with the prolongation of differentiation time; Significantly promoted the expression of γ-globin and β-globin at the mRNA level.

Example 3: Western Blot experiment

The obtained cells were collected and lysed at different time points according to conventional methods, and the protein was extracted and subjected to western blot analysis to detect the expression changes of erythroid marker molecules γ-globin and β-globin.

The antibodies used in the experiment are shown in Table 2:

Table 2

The results are shown in Figure 2. When HSPCs were induced to differentiate, the expression of erythroid marker molecules γ-globin and β-globin at the protein level gradually increased with the prolongation of differentiation time; Significantly promoted the expression of γ-globin and β-globin at the protein level.

Example 4: Flow Cytometry

Cells were collected at different time points according to conventional methods, incubated in APC-anti-CD235a/FITC-anti-CD71 antibody mixture for 30 min (room temperature, shaker), and then detected the expression of CD71/CD235a on the machine. Both CD71 and CD235a are membrane surface markers of erythroid cells, and the ratio of CD71 ⁺ /CD235a ⁺ cells represents the degree of erythroid cell differentiation. FITC-anti-CD71 antibody (Cat. No.: 555536) was purchased from BD Biosciences, USA, and APC-anti-CD235a (Cat. No. 17-9987-41) was purchased from Thermo Fisher Scientific, USA.

The results of flow cytometry experiments showed that the ratio of CD71 ^{+ /CD235a + cells gradually increased with the process of differentiation, and the ratio of CD71 + /CD235a + cells in} the hypoxia experimental group was significantly higher than that in the normoxia group (Figure 3).

Example 5:

Cells were collected at different time points according to conventional methods, centrifuged on glass slides, and stained according to the instructions of Giemsa Staining Kit (Cat. No.: G1021) to observe cell morphological changes. The Giemsa staining kit was purchased from Solarbio, China.

Figure 4 shows the statistical results of the morphological changes of the induced erythroid cells after being cultured under normoxia (Nor) or hypoxia (Hyp) for different times. For example, on the 11th day of induction of differentiation, the proportion of mature erythrocytes in the hypoxia group was significantly higher in the normoxic group.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (8)

1. A method of promoting differentiation of human hematopoietic stem/progenitor cells into erythroid cells, said method comprising administering to a human hematopoietic stem/progenitor cell culture a hypoxic environment or a drug that mimics a hypoxic environment.

2. The method of promoting erythroid differentiation of human hematopoietic stem/progenitor cells according to claim 1, wherein said hypoxic environment is an oxygen content of less than 3%.

3. The method of promoting erythroid differentiation of hematopoietic stem/progenitor cells according to claim 1, wherein the agent that mimics a hypoxic environment is FG-4592, COCl₂And one or more of deferoxamine.

4. The method of promoting differentiation of hematopoietic stem/progenitor cells into erythroid cells according to claim 1, wherein the agent that mimics a hypoxic environment further comprises one or more pharmaceutically acceptable excipients or carriers.

5. The method for promoting differentiation of hematopoietic stem/progenitor cells into erythroid cells according to claim 4, wherein the adjuvant or carrier comprises one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, and lubricants, which are conventional in the pharmaceutical field.

6. The method for promoting differentiation of hematopoietic stem/progenitor cells into erythroid cells according to claim 1, wherein the agent simulating hypoxic environment is formulated into a dosage form selected from the group consisting of tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral liquids, pills and injections.

7. The method of promoting the differentiation of human hematopoietic stem/progenitor cells into erythroid lineage according to claim 1, wherein the hematopoietic stem/progenitor cells are derived from umbilical cord blood, bone marrow or peripheral blood.

8. Use of erythrocytes obtained according to the process of claim 1, for the preparation of a medicament for the treatment of wounds, hematological disorders, immunodeficiency syndrome, malignancies, anemia.

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